LOCUS       sequence-469887-        6045 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the mCherry-based sensor for miR-124. TagBFP2 is expressed
            as a transduction reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6045)
  AUTHORS   Zolboot N, Xiao Y, Du JX, Ghanem MM, Choi SY, Junn MJ, Zampa F, 
            Huang Z, MacRae IJ, Lippi G
  TITLE     MicroRNA mechanisms instructing Purkinje cell specification.
  JOURNAL   Neuron. 2025 Apr 1:S0896-6273(25)00181-3. doi: 
            10.1016/j.neuron.2025.03.009.
  PUBMED    40179877
REFERENCE   2  (bases 1 to 6045)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Neuron. 
            2025 Apr 1:S0896-6273(25)00181-3. doi: 
            10.1016/j.neuron.2025.03.009."
COMMENT     Sequence Label: pAAV-rev(CMV-TagBFP2)-EFS-mCherry-124-WT-MRE
FEATURES             Location/Qualifiers
     source          1..6045
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     protein_bind    148..168
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    297..431
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(334..353)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(467..1168)
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    1182..1206
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(1228..1431)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1257..1277)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1432..1735
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    1811..1902
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(1843..1862)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    1916..1964
                     /note="synthetic polyadenylation signal"
     protein_bind    1965..1985
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        1992..2203
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2228..2237
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2234..2944
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2379..2397)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2647..2666
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3087..3104)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3093..3300
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3308..3448
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3308..3437
                     /label=AAV2 ITR
     rep_origin      3523..3978
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3610..3629)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3820..3841
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3995..4014)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4114..4136
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4174..4192)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4260..4364
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4365..4433,4434..5225)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4583..4602)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5396..5984
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5885..5904
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc ttctagacaa ctttgtatag aaaagttgcc cttaattaat
      181 cgagagatct gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
      241 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
      301 tgtttattgc agcttataat ggttacaaat aaggcaatag catcacaaat ttcacaaata
      361 aggcattttt ttcactgcat tctagttttg gtttgtccaa actcatcaat gtatcttatc
      421 atgtctggat ctcgcggccg cactatgtat aataaagttg ggtacctcaa ttaagcttgt
      481 gccccagttt gctagggagg tcgcagtatc tggccactgc cacctcgtgc tgctcgacgt
      541 aggtctcgtt gttggcctcc ttgattcttt ccagtctgta gtccacatag tagacgccag
      601 gcatcttgag gttcttagcg ggtttcttgg atctatatgt ggtcttggcg tttgcgatca
      661 gatggctccc gcccacgagc ttcagggcca tgtcgtttct gccttccagg ccgccgtcag
      721 cggggtacag cgtctcggtg aaggcctccc agccgagtgt tttcttctgc atcacagggc
      781 cgttggatgt gaagttcacc cctctgatct tgacgttgta gatgaggcag ccgtcctgga
      841 ggctggtgtc ctgggtagcg gtcagcacgc ccccgtcttc gtatgtggtg actctctccc
      901 atgtgaagcc ctcagggaag gactgcttga agaagtcggg gatgccctgg gtgtggttga
      961 tgaaggtctt gctgccgtag aggaagctag tagccaggat gtcgaaggcg aaggggagag
     1021 ggccgccctc gaccaccttg attctcatgg tctgggtgcc ctcgtagggc ttgccttcgc
     1081 cctcggatgt gcacttgaag tgatggttgt ccacggtgcc ctccatgtac agcttcatgt
     1141 gcatgttctc cttaatcagc tcgctcatgg tggcggcgcg ccccactttg tacaagaaag
     1201 ctgggtgatc tgacggttca ctaaaccagc tctgcttata tagacctccc accgtacacg
     1261 cctaccgccc atttgcgtca atggggcgga gttgttacga cattttggaa agtcccgttg
     1321 attttggtgc caaaacaaac tcccattgac gtcaatgggg tggagacttg gaaatccccg
     1381 tgagtcaaac cgctatccac gcccattgat gtactgccaa aaccgcatca ccatggtaat
     1441 agcgatgact aatacgtaga tgtactgcca agtaggaaag tcccataagg tcatgtactg
     1501 ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat
     1561 atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg
     1621 tcaatggaaa gtccctattg gcgttactat gggaacatac gtcattattg acgtcaatgg
     1681 gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa
     1741 ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactattaat aactactcga
     1801 gagcctgctt agagaaatgt tctggcacct gcacttgcac tggggacagc ctattttgct
     1861 agtttgtttt gtttcgtttt gttttgatgg agagcgtatg ttagtactat cgattcacac
     1921 aaaaaaccaa cacacagatg taatgaaaat aaagatattt tattcaactt tgtatagaaa
     1981 agttgactag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
     2041 cggcaattga tccggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
     2101 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
     2161 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggcgtacg gccaccatga
     2221 gaccggtgcc accatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     2281 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     2341 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     2401 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     2461 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     2521 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     2581 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     2641 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     2701 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     2761 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     2821 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     2881 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     2941 gtaacaactt ggatccagtg tggaggactt aatcggtcat ttgctcacta tggaatgtgg
     3001 tctcttctgc tttgtgcctt agttttttaa tctgttcccc tttgaataca gaatccacgc
     3061 gtgaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     3121 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     3181 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     3241 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga
     3301 gggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     3361 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     3421 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     3481 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     3541 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
     3601 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3661 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3721 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     3781 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     3841 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     3901 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     3961 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4021 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     4081 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     4141 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     4201 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     4261 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4321 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4381 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4441 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4501 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     4561 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     4621 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     4681 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     4741 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     4801 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     4861 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     4921 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     4981 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5041 gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag
     5101 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5161 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5221 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5281 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5341 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5401 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5461 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5521 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     5581 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5641 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5701 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5761 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5821 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5881 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5941 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6001 cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
